2.1. EXOGENOUS GENE EXPRESSION
Advances in recombinant DNA technology have permitted the expression of exogenous foreign genes in eukaryotic cells. Foreign gene expression has not only facilitated studies of gene function and the production of properly processed gene products, it has also led to the possibility of correcting gene defects by introducing functionally active genes in gene therapy.
A number of gene delivery and expression systems have been developed and successfully used to express foreign genes in eukaryotic cells, and less satisfactorily, in animals. These systems consist primarily of a DNA delivery method and a DNA expression vector. DNA delivery methods include calcium phosphate precipitation (Wigler et al., 1979, Proc. Natl. Acad. Sci. USA 76:1373-1376), DEAE-dextran (Sompagnac et al., 1981, Proc. Natl. Acad. Sci. USA 78:7575-7578), lipofection (Felgner et al., 1987, Proc. Natl. Acad. Sci. USA 84:7413-7417), electroporation (Neumann et al., 1982, EMBO J. 7:841-845), retroviruses (Schimotohono et al., 1981, Cell 26:67-77), direct DNA injection (Benvensty et al., 1986, Proc. Natl. Acad. Sci. USA 83:9551-9555; Wolff et al., 1990, Science 247:1465-1468), specific receptor-mediated DNA uptake (Wu et al., 1988, J. Biol. Chem. 263:14621-14624; Wu et al., 1989, J. Biol. Chem. 264:16985-16987), and more recently, aerosol DNA delivery (Stribling et al., 1992, Proc. Natl. Acad. Sci. USA 89:11277-11281). Expression vectors are in general plasmid DNA containing a gene of interest linked to a regulatory sequence which controls the expression of the gene upon its introduction into a given cell.
Since these gene expression systems usually utilize eukaryotic or mammalian promoters to direct gene transcription, they require entry into host cell nuclei and integration of the exogenous DNA for expression. However, only a very small percentage of the cells which take up DNA contain the foreign DNA within the nuclei of the cells. This problem becomes even more pronounced when whole animals are the targets of foreign gene expression because cell division, which is required for nuclear deposition of the introduced DNA, does not always occur at as high a frequency in the cells of tissues as it does in cultured cell lines, making it difficult for exogenous DNA to integrate into non-dividing cells.
On the other hand, evidence has indicated that the cytoplasmic uptake of foreign DNA by cells and animals is efficient (Felgner et al., 1987, Proc. Natl. Acad. Sci. USA 84:7413-7417). And, in certain circumstances, the DNA introduced into the cells of whole animal tissues has been observed to remain in the cytoplasm up to several months (Wolff et al., 1990, Science 247:1465-1468).
2.2. BACTERIOPHAGE RNA POLYMERASE
Bacteriophage T7 RNA polymerase (RNAP) has been studied extensively in vitro and in vivo in bacteria and eukaryotic cells due to several of its unique biochemical characteristics. In particular, T7 RNAP is a single polypeptide enzyme capable of carrying out transcription with high promoter specificity and efficiency, without the involvement of any other cellular transcription factors (Davanloo et al., 1984, Proc. Natl. Acad. Sci. USA 81:2035-2039; Dunn et al., 1983, J. Mol. Biol. 166:477-535; Studier et al., 1990, Methods Enzymol. 185:60-89). For example, recombinant vaccinia viruses containing a T7 RNAP gene or a cell line which constitutively expresses T7 RNAP have been used to promote expression of genes of interest linked to T7 promoter in the cytoplasm of mammalian cells (Fuerst et al., 1986, Proc. Natl. Acad. Sci. 83:8122-8126; Fuerst et al., 1989, J. Mol. Biol. 206:333-348; Elroy-Stein et al., 1989, Proc. Natl. Acad. Sci. USA 86:6126-6130; Elroy-Stein et al., 1990, Proc. Natl. Acad. Sci. USA 87: 6743-6747). When a chloramphenicol acetyltransferase (CAT) gene was inserted into a T7 promoter-containing mammalian vector and transfected into a stable cell line which also expressed T7 RNAP, as high as 30% of cytoplasmic proteins was found to be the CAT enzymes in the transfected cells (Elroy-Stein et al., 1990, Proc. Natl. Acad. Sci. USA 87: 6743-6747). However, a major limitation of such a T7 gene expression system is that it must be performed in cell lines which also express the T7 RNAP gene, which is a bacterial gene not expressed endogenously in eukaryotic cells.